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Kostas Tokatlidis University of Crete and IMBB-FORTH

Oxidative folding in mitochondria: From electron transfer reactions to cellular architecture and disease. Kostas Tokatlidis University of Crete and IMBB-FORTH. 1988, BSc, Chemical Engineering, Aristotle Univ. Thessaloniki 1991, MChem, Chemical Engineering, Univ of Delaware, USA

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Kostas Tokatlidis University of Crete and IMBB-FORTH

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  1. Oxidative folding in mitochondria: From electron transfer reactions to cellular architecture and disease Kostas Tokatlidis University of Crete and IMBB-FORTH

  2. 1988, BSc, Chemical Engineering, Aristotle Univ. Thessaloniki 1991, MChem, Chemical Engineering, Univ of Delaware, USA 1993, PhD, (Fulbright and EU Fellow) Chemical Engineering/Biochemistry Univ. of Delaware and Institut Pasteur France 1993-1994, postdoc (EU Fellow), Institut Pasteur France 1994-1998, postdoc (HFSP, EMBO, Roche Fellow) Biozentrum, Basel, Switzerland 1998-2003, Lecturer, Senior Lecturer, Lister Fellow, Univ. Manchester, UK 2003- now, Group Leader, IMBB-FORTH 2003-2006, Assistant Prof, Dept Chemistry, UoC 2007-now, Associate Prof, Dept Materials Science and Technology, UoC

  3. Drug 1900s Ehrlich , ‘Magic bullet’ Goal: Tailored and efficient therapeutics Critical need for drug delivery site-specifically at the subcellular and suborganellar Level ? ?

  4. The Biological Problem • Mito facts: • 1500 proteins • own mtDNA encoding only 13 proteins • >99% have to be imported • Protein import is the crucial mechanism ofmitochondria biogenesis 1. Components? 2. Mechanisms? 3. Relevance in health and disease? • 90% of the cells energy is provided • by mitochondria • More than 300 mitochondrial diseases • Involved in ageing, cancer, heart disease • Key regulators of apoptosis • United Mitochondrial Disease foundation: a child born every 15 min suffers or • will develop a mito disease by the age of 5

  5. more than 30% of proteome are membrane proteins About 50% of drug targets in Pharma Industry are membrane proteins

  6. Mitochondria are essential for life

  7. Functional Complexity Respiration and ATP Synthesis Synthesis of heme, lipids, amino acids and nucleotides Intracellular homeostasis of inorganic ions Structural Complexity 5-15% of total cell protein 20% volume of eukaryotic cell IM is 1/3 of total cell membrane About 1000 different polypeptides (900 in yeast) Only a dozen encoded by mtDNA

  8. Protein import is the major mechanism of mitochondria biogenesis Curiosity-driven research: How do proteins find their way to mitochondria?

  9. Approach Isolated molecules In vitro Intact cells In vivo Isolated organelles In organello

  10. Lithgow and Pfanner, 2005

  11. By using … Protein purification Mutagenesis CD analysis Limited proteolysis Bioinformatics Mass spectrometry Chemical modification of thiol groups. in vivo thiol trapping Gel filtration Isothermal titration calorimetry ITC Analytical centrifugation Multi-angle light static scattering

  12. NOVEL oxidative folding pathway operating in mitochondria in vivo

  13. …closing the loop: CytC and the respiratory chain are the final acceptors of electrons from the imported precursor Allen et al., JMB, 2005; cover

  14. Functional and Structural analysis of Mia40

  15. Solution structure of ΜΙΑ40 byNMR Banci et al., Nature SMB, 2009

  16. The hydrophobic cleft mediates non-covalent binding of the substrate Banci et al., Nature SMB, 2009

  17. Structural basis for the binding of the ITSonto the cleft of Mia40 Sideris et al. 2009 JCB

  18. Mechanism of substrate recognition by Mia40: The sliding – docking model

  19. Conclusions An oxidative folding pathway operates in mitochondria Docking of the substrate to the Mia40 represents a site specific event that is crucial step for the oxidative folding process The process is guided by a novel ITS that directs the first step of noncovalent recognition by Mia40 Mia40 represents structurally, functionally and mechanistically a new type of cellular oxidoreductase Selective publications 2009-2011 Nature Structural and Molecular Biology 16(2), 198-206, 2009 J. Cell Biol. 187(7), 1007-1022, 2009 Proc Natl Acad. Sci USA107(47), 20190-20195, 2010 Proc Natl Acad. Sci USA108(12), 4811-4816,2011

  20. Future Directions: • - Mechanism of protein-protein interactions and structural basis • - Links to cytochrome C mediated cell-death pathways • Other substrates of Erv1 (ALS-linked SOD1, lifespan/aging determinants • targeted to the IMS)

  21. A new mechanism of peptide-based targeting in the oxidative folding pathway in mitochondria

  22. Acknowledgements • IMBB/UoCrete: • Afroditi Hatzi (PhD) • Emanouela Kalergi (PhD) • Maria Andreadaki (MSc) • Nitsa Katrakili (BSc Chem Eng.) • Alumni from Crete: • - Dionisia(Jenny) Sideris (PhD 2009, postdoc Biology/MIT) • Eirini Lionaki (PhD student) Nikos Petrakis (PhD 2009, postdoc MAICH) • Paraskevi Kritsiligkou (Diploma 2009 MSc Biochemistry/ Oxford) • - Fliss Alcock (PhD 2008, • postdoc Monash, Australia) • Vasilia Balabanidou (MSc 2005, PhD IMBB) • Carine de Marcos (postdoc, Leeds UK) • - Catherine Baud (postdoc, Toulouse France) Funding: GSRT, IMBB, UoC EU NMR, EU RegPot Collaborators CERM-Florence (NMR)

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